ANTI-HUMAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1 (hENT1) ANTIBODIES ADN METHODS OF USE THEREOF

ABSTRACT

This invention provides monoclonal antibodies that recognize hENT1. The invention further provides methods of using such monoclonal antibodies as a therapeutic, diagnostic, and/or prophylactic in disorders associated with aberrant hENT1 expression and/or activity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/161,941, filed Jun. 16, 2011, which claims the benefit of U.S.Provisional Application No. 61/355,357, filed Jun. 16, 2010 and U.S.Provisional Application No. 61/432,702, filed Jan. 14, 2011. Thecontents of each of these applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of monoclonalantibodies that recognize human Equilibrative Nucleoside Transporter 1(hENT1), and to methods of using these monoclonal antibodies astherapeutics.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “37269503C01US_SeqList.txt,” whichwas created on May 12, 2014 and is 61.0 KB in size, are herebyincorporated by reference in their entirety

BACKGROUND OF THE INVENTION

Human equilibrative nucleoside transporter 1 (hENT1) is a protein thatis encoded by the SLC29A1 gene. This gene is a member of theequilibrative nucleoside transporter family. The gene encodes atransmembrane glycoprotein that localizes to the plasma andmitochondrial membranes and mediates the cellular uptake of nucleosidesfrom the surrounding medium. The protein is categorized as anequilibrative (as opposed to concentrative) transporter that issensitive to inhibition by nitrobenzylthioinosine (NBMPR). Nucleosidetransporters are required for nucleotide synthesis in cells that lack denovo nucleoside synthesis pathways, and are also necessary for theuptake of cytotoxic nucleosides and nucleoside analogue drugs used forcancer and viral chemotherapies.

Accordingly, there exists a need for therapies that treat or otherwiseameliorate a disorder associated with aberrant hENT1 expression and/oractivity.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies which specificallybind to human Equilibrative Nucleoside Transporter 1 (hENT1) or abiologically active fragment thereof. The hENT1 antibodies are useful indetecting patients or patient samples with low or otherwise reducedhENT1 expression and/or activity. For example, the hENT1 antibodies canbe used to screen patients having cancers and other neoplastic disordersin which hENT1 expression and/or activity is low or otherwise reduced,such as, for example, acute myeloid leukemia (AML) or pancreatic cancerand other solid tumors. The hENT1 antibodies are useful to identifypatients for treatment based on detected hENT1 expression and/oractivity level. The hENT1 antibodies are useful as a prognosis tool forsubjects diagnosed a disorder associated with low hENT1 expressionand/or activity such as, for example, cancer or other neoplasticdisorders. Prognosis of a disorder associated with low hENT1 expressionand/or activity is also determined by measuring hENT1 expression and/oractivity level over time, such as for example, during the course oftherapeutic methods.

The hENT1 antibodies of the invention are useful as companiondiagnostics for established cancer drugs such as nucleoside analoguedrugs and/or drugs derived from nucleoside analogues. For example, thehENT1 antibodies are useful in conjunction with chemotherapy agents suchas pyrimidine derivatives including, for example, cytarabine,gemcitabine, azacytidine, and derivatives thereof, and purinederivatives including, for example, fludarabine, cladribine, clofarabineand derivatives thereof.

Exemplary monoclonal antibodies of the invention include, for example, avariable heavy chain sequence such the VH3-12 variable heavy chain, theVH5-9 variable heavy chain, the VH5-12 variable heavy chain, the VH5-13variable heavy chain, the VH5-3 variable heavy chain, the VH5-11variable heavy chain, or the consensus variable heavy chain providedherein and referred to as the consensus variable heavy chain regionsequence 1 (consensus VH sequence 1). Exemplary variable heavy chainsequences for the hENT1 antibodies of the invention also include theVH1-1 variable heavy chain, the VH1-4 variable heavy chain, the VH1-6variable heavy chain, the VH4-2 variable heavy chain, the VH4-3 variableheavy chain, the VH4-4 variable heavy chain or the consensus variableheavy chain provided herein and referred to as the consensus variableheavy chain region sequence 2 (consensus VH sequence 2). Exemplarymonoclonal antibodies of the invention include, for example, a lightchain variable sequence such as, for example, the VL2 variable lightchain, the VL10 variable light chain, the VL11 variable light chain, theVL20 variable light chain, the VL21 light chain, or the consensusvariable light chain provided herein and referred to as the consensusvariable light chain region sequence 1 (consensus VL sequence 1).Exemplary variable light chain sequences for the hENT1 antibodies of theinvention also include the VL2-2 variable light chain, the VL2-3variable light chain, the VL2-7 variable light chain, the VL2-10variable light chain, the VL2-12 variable light chain, the VL2-16variable light chain or the consensus variable light chain providedherein and referred to as the consensus variable light chain regionsequence 2 (consensus VL sequence 2). These antibodies are respectivelyreferred to herein as “hENT1 antibodies” or “anti-hENT1 antibodies”.hENT1 antibodies include fully human monoclonal antibodies, as well ashumanized monoclonal antibodies and chimeric antibodies. Theseantibodies show specificity for hENT1.

The hENT1 antibodies of the invention include a heavy chain variableregion having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 9, 28,30, 32, 34, 36, 38 or 39. The hENT1 antibodies of the invention includea light chain variable region having the amino acid sequence of SEQ IDNOs: 14, 16, 18, 20, 22, 23, 43, 45, 47 or 49.

Preferably, the variable heavy chains of the hENT1 antibodies of theinvention include a variable heavy chain complementarity determiningregion 1 (VH CDR1) sequence comprising the amino acid sequence GYTFTDYE(SEQ ID NO: 10), a variable heavy chain complementarity determiningregion 2 (VH CDR2) sequence comprising the amino acid sequence IDPETGAI(SEQ ID NO: 11) or the amino acid sequence IDPETGKT (SEQ ID NO: 40), anda variable heavy chain complementarity determining region 3 (VH CDR3)sequence comprising the amino acid sequence TREFTY (SEQ ID NO: 12) orthe amino acid sequence TRELTY (SEQ ID NO: 41).

The heavy chain variable regions of the hENT1 antibodies of theinvention contain a variable heavy chain complementarity determiningregion 1 (VH CDR1) that includes an amino acid sequence at least 90%,92%, 95%, 97% 98%, 99% or more identical to the amino acid sequenceGYTFTDYE (SEQ ID NO: 10); a variable heavy chain complementaritydetermining region 2 (VH CDR2) that includes an amino acid sequence atleast 90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acidsequence IDPETGAI (SEQ ID NO: 11) or the amino acid sequence IDPETGKT(SEQ ID NO: 40); and a variable heavy chain complementarity determiningregion 3 (VH CDR3) that includes an amino acid sequence at least 90%,92%, 95%, 97% 98%, 99% or more identical to the amino acid sequenceTREFTY (SEQ ID NO: 12) or the amino acid sequence TRELTY (SEQ ID NO:41).

The present invention provides methods of detecting hENT1 activityand/or expression in a patient or patient sample. The invention providesuses of the hENT1 antibodies in conjunction with establishedchemotherapies including, for example, nucleoside analogue drugs and/ordrugs derived from nucleoside analogues. For example, the hENT1antibodies are used in conjunction with chemotherapy agents such aspyrimidine derivatives including, for example, cytarabine, gemcitabine,azacytidine, and derivatives thereof, and purine derivatives including,for example, fludarabine, cladribine, clofarabine and derivativesthereof. Other suitable agents for use in combination with the hENT1antibodies disclosed herein include antiviral agents such as, forexample, ribavirin, and anticancer nucleoside agents such as5-fluorouridine, which are substrates of nucleoside transporters. Thus,the hENT1 antibodies are also useful in conjunction with antiviraltherapies, anticancer therapies and derivatives thereof.

The present invention also provides methods of treating or preventingpathologies associated with aberrant hENT1 expression and/or activity,or alleviating a symptom associated with such pathologies, byadministering a monoclonal antibody of the invention to a subject inwhich such treatment or prevention is desired. The terms “patient” and“subject” are used interchangeably herein and throughout thisdescription. The subject to be treated is, e.g., human. The monoclonalantibody is administered in an amount sufficient to treat, prevent oralleviate a symptom associated with the pathology. The amount ofmonoclonal antibody sufficient to treat or prevent the pathology in thesubject is, for example, an amount that is sufficient to reduce hENT1expression and/or activity.

The antibodies of the invention are capable of modulating, e.g.,blocking, inhibiting, reducing, antagonizing, neutralizing or otherwiseinterfering with expression of hENT1 and/or one or more biologicalactivities of hENT1. For example, the hENT1 antibodies completely orpartially inhibit hENT1 expression and/or biological activity bypartially or completely modulating, blocking, inhibiting, reducingantagonizing, neutralizing, or otherwise interfering with the expressionand/or activity of hENT1, or otherwise partially or completelymodulating, blocking, inhibiting, reducing, antagonizing, neutralizinghENT1 expression and/or activity. The hENT1 antibodies are considered tocompletely modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with hENT1 expression and/or biological activitywhen the level of hENT1 expression and/or activity in the presence ofthe hENT1 antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%,99% or 100% as compared to the level of hENT1 expression and/or activityin the absence of binding with an hENT1 antibody described herein. ThehENT1 antibodies are considered to partially modulate, block, inhibit,reduce, antagonize, neutralize or otherwise interfere with hENT1expression and/or activity when the level of hENT1 expression and/oractivity in the presence of the hENT1 antibody is decreased by less than95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% ascompared to the level of hENT1 expression and/or activity in the absenceof binding with an hENT1 antibody described herein.

As used herein, the term “reduced” refers to a decreased expressionand/or activity of hENT1 in the presence of a monoclonal antibody of theinvention. hENT1 expression is decreased when the level of hENT1expression in the presence of a monoclonal antibody of the invention isgreater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 90%, 95%, 99%, or 100% lower than a control level of hENT1expression. hENT1 expression level is determined using a variety ofassays, including those described in the Examples provided herein. hENT1activity is decreased when the level of one or more biologicalactivities of hENT1 in the presence of a monoclonal antibody of theinvention is greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% lower than a control level ofhENT1 activity. hENT1 activity level is determined using a variety ofassays.

Pathologies treated, ameliorated and/or prevented using the monoclonalantibodies of the invention (e.g., monoclonal antibodies) include, forexample, cancer and other neoplastic indications such as AML andpancreatic cancer. Pathologies treated, ameliorated and/or preventedusing the monoclonal antibodies of the invention (e.g., monoclonalantibodies) include, for example, viral indications such as hepatitis C,infectious diseases such as malaria, and blood diseases such asbeta-thalassemia.

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the expression vector used toexpress the anti-hENT1 antibodies.

FIG. 2 is a graph depicting the results of a quantitative ELISA assayusing standard murine IgG.

FIG. 3 is an illustration depicting the results of non-reducing westernblot analysis of unconcentrated supernatant and positive control murineIgG after 1 min exposure.

FIG. 4 is an illustration depicting the results of non-reducing westernblot analysis, where the blot was over-developed to show positive bandsin the supernatants after 5 min exposure.

FIG. 5 is a schematic representation of the expression vector used toexpress additional anti-hENT1 antibodies.

DETAILED DESCRIPTION

The present invention provides monoclonal antibodies that specificallybind hENT1. The antibodies of the present invention bind to an hENT1epitope with an equilibrium binding constant (K_(d)) of ≦1 μM, e.g.,≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM. For example, thehENT1 antibodies provided herein exhibit a K_(d) in the rangeapproximately between ≦1 nM to about 1 pM.

Nucleoside analogue drugs depend on nucleoside transporters to enter thecells where they exert their effect—these molecules do not cross theplasma membrane by diffusion, and efficient cellular uptake requires thepresence of these specialized plasma membrane nucleoside transporterproteins.

Cytarabine and gemcitabine (Gemzar, Eli Lilly, Indianapolis, Ind.) aretwo anticancer drugs that depend on human Equilibrative NucleosideTransporter 1 (hENT1) for their effect. A considerable proportion ofcancer cells have low expression of hENT1. Low expression and/oractivity of hENT1 has been found in patients having cancers and otherneoplastic disorders such as, for example, acute myeloid leukemia (AML)or pancreatic cancer. Low clinical effect of treatment has beencorrelated with reduced or no presence of hENT1 in the cancer cells.(See e.g., Galmarini, et al., “Potential mechanisms of resistance tocytarabine in AML patients,” Leuk Res 26 (2002) 621-629; Farrell, etal., “Human Equilibrative Nucleoside Transporter 1 Levels PredictResponse to Gemcitabine in Patients With Pancreatic Cancer,”Gastroenterology 136 (2009) 187-195; and Giovannetti, et al.,“Transcription analysis of human equilibrative nucleoside transporter-1predicts survival in pancreas cancer patients treated with gemcitabine,”Cancer Res 66 (2006) 3928-3935). Studies have shown a direct correlationbetween the lack of hENT1 expression on a pancreatic cancer patient'stumor cells and that patient's poor response to nucleoside analogue andother chemotherapeutic drugs such as gemcitabine. (See Farrell, et al.,“Human Equilibrative Nucleoside Transporter 1 Levels Predict Response toGemcitabine in Patients With Pancreatic Cancer,” Gastroenterology 136(2009) 187-195). hENT1 is also important for the oral uptake ofribavirin and can cause resistance to ribavirin hepatitis C treatment.(See Ibarra et al., “Reduced ribavirin antiviral efficacy via nucleosidetransporter-mediated drug resistance,” J. Virol., vol. 83(9): 4583-47(2009)).

Thus, the hENT1 antibodies of the invention are useful in detectinghENT1 levels in a patient or patient sample. The hENT1 antibodies of theinvention are useful as companion diagnostics for current cancer drugsor antiviral drugs. For example, the hENT1 antibodies are useful inconjunction with chemotherapy agents such as nucleoside analogue drugsincluding cytarabine, azacytidine gemcitabine, gemcitabine-5′-elaidicacid ester, cytarabine-5′-elaidic acid ester, 5-azacytidine-5′-elaidicacid ester and ribavirin-5′-elaidic acid ester. The hENT1 antibodies arealso useful in combinational with agents such as purine and pyrimidinenucleoside derivatives. (See Quashie et al., “Uptake of purines inPlasmodium falciparum-infected human erythrocytes is mostly mediated bythe human equilibrative nucleoside transporter and the humanfacilitative nucleobase transporter,” Malaria Journal, vol. 9: 36(2010)).

The hENT1 antibodies of the invention are useful to identify apopulation of patients with low or otherwise reduced hENT1 expressionand/or activity for additional or otherwise altered treatment regimens.For example, this identified patient population is administered atreatment that is designed to allow uptake in hENT1 deficient cells,such as for example, gemcitabine-5′-elaidic acid ester orgemcitabine-N4-elaidic acid amide, cytarabine-5′-elaidic acid ester, or5-azacytidine-5′-elaidic acid ester or ribavirin-5′-elaidic acid ester.(See Brueckner, et al., “Delivery of 5-azacytidine to human cancer cellsby elaidic acid esterification increases therapeutic drug efficacy,” MolCancer Ther., vol. 9(5): 1256-1264 (2010)). Studies have shown thatthese drugs, in contrast to established nucleoside drugs such asgemcitabine, cytarabine and azacytidine, are able to enter cancer cellsand retain their activity in cancer cells independent of the hENT1expression level in the cancer cell. (See e.g. Breistol, et al.,“Antitumor activity of P-4055 (elaidic acid-cytarabine) compared tocytarabine in metastatic and s.c. human tumor xenograft models,” CancerRes 59 (1999) 2944-2949; and Galmarini et al., “CP-4055 and CP-4126 areactive in ara-C and gemcitabine-resistant lymphoma cell lines,” Br JHaematol 144 (2009) 273-275).

The uptake of gemcitabine-5′-elaidic acid ester andcytarabine-5′-elaidic acid ester in hENT1 deficient cells has beenconfirmed also in vitro with confirmed high formation of the activetriphosphate metabolite of cytarabine-5′-elaidic acid ester andgemcitabine-5′-elaidic acid ester in deficient cancer cells. Once insidethe cell the lipid tail of the lipid-conjugated drug such asgemcitabine-5′-elaidic acid ester or cytarabine-5′-elaidic acid ester iscleaved off, and the parent drug is released. With the lack of hENT1transporter (due to the low expression and/or activity of hENT1 in theidentified patient population), the drug is trapped inside the cell, andhigh concentrations of active metabolites have been measured. (See Ademaet al., “Fatty acid derivatives of cytarabine and gemcitabine, CP-4055and CP-4126, show a prolonged cellular retention compared to the parentdrug,” Proceedings of the 99th Annual Meeting of the AmericanAssociation for Cancer Research. Abstract nr 5740 (2008).

These observations indicate that lipid-conjugated gemcitabinederivatives such as gemcitabine-5′-elaidic acid ester and/orlipid-conjugated cytarabine derivatives such as cytarabine-5′-elaidicacid ester are useful in treating tumors that are resistant or otherwiseless responsive to cytarabine and gemcitabine due to the lack of hENT1or low hENT1 expression and/or activity. Thus, the antibodies of theinvention are useful in combination therapies with theselipid-conjugated gemcitabine derivatives such as gemcitabine-5′-elaidicacid ester and/or lipid-conjugated cytarabine derivatives such ascytarabine-5′-elaidic acid ester and/or lipid-conjugated as5-azacytidine-5′-elaidic acid ester. For example, the hENT1 antibodiesare useful in detecting patients with low or otherwise reduced hENT1expression and/or activity. For example, the hENT1 antibodies can beused to screen patients having cancers and other neoplastic disorderssuch as acute myeloid leukemia (AML) or pancreatic cancer. Thus, thehENT1 antibodies are useful to identify patients for treatment based onthe hENT1 expression and/or activity level.

In some embodiments, the patients are currently receiving treatmentregimens that include administration of one or more nucleoside analoguedrugs and/or drugs derived from nucleoside analogues, such as,pyrimidine derivatives including, for example, cytarabine, gemcitabine,and derivatives thereof, and purine derivatives including, for example,fludarabine, cladribine, clofarabine and derivatives thereof. In someembodiments, the patients are currently receiving treatment regimensthat include administration of one or more nucleoside analogue drugsand/or drugs derived from nucleoside analogues, such as pyrimidinederivatives including, for example, cytarabine, gemcitabine, andderivatives thereof d purine derivatives including, for example,fludarabine, cladribine, clofarabine and derivatives thereof, and thesepatients have stopped responding to treatment or are otherwise lessresponsive to the nucleoside analogue drug.

In some embodiments, the patients have previously received treatmentregimens that included administration of one or more nucleoside analoguedrugs and/or drugs derived from nucleoside analogues, such as pyrimidinederivatives including, for example, cytarabine, gemcitabine, andderivatives thereof and purine derivatives including, for example,fludarabine, cladribine, clofarabine and derivatives thereof. In someembodiments, the patients have previously received treatment regimensthat included administration of one or more nucleoside analogue drugsand/or drugs derived from nucleoside analogues, such as pyrimidinederivatives including, for example, cytarabine, gemcitabine, andderivatives thereof and purine derivatives including, for example,fludarabine, cladribine, clofarabine and derivatives thereof, and thesepatients stopped responding to treatment or were otherwise lessresponsive to the nucleoside analogue drug.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms “human Equilibrative Nucleoside Transport 1”and “hENT1” are synonymous and may be used interchangeably.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat et al., Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al.Nature 342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 μM;e.g., ≦100 nM, preferably ≦10 nM and more preferably ≦1 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” (and all grammatical variationsthereof) refer to the non-covalent interactions of the type which occurbetween an immunoglobulin molecule and an antigen for which theimmunoglobulin is specific. The strength, or affinity of immunologicalbinding interactions can be expressed in terms of the dissociationconstant (K_(d)) of the interaction, wherein a smaller K_(d) representsa greater affinity. Immunological binding properties of selectedpolypeptides can be quantified using methods well known in the art. Onesuch method entails measuring the rates of antigen-binding site/antigencomplex formation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to hENT1, when the equilibrium bindingconstant (K_(d)) is ≦1 μM, preferably 100 nM, more preferably ≦10 nM,and most preferably ≦100 pM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

The term “biological sample” is intended to include tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component ofblood including blood serum, blood plasma, or lymph.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules presented in SEQ IDNOS: 1, 3, 5, 7, 27, 29, 31, 33, and 37, and nucleic acid moleculesencoding the light chain immunoglobulin molecules represented in SEQ IDNOS: 13, 15, 17, 21, 42, 44, 46 and 48.

hENT1 Antibodies

Monoclonal antibodies of the invention bind hENT1. These monoclonalantibodies have the ability to inhibit hENT1 expression and/or activityInhibition is determined, for example, using any of a variety ofstandard assays.

Exemplary variable heavy chain sequences for the hENT1 antibodies of theinvention include the VH3-12 variable heavy chain, the VH5-9 variableheavy chain, the VH5-12 variable heavy chain, the VH5-13 variable heavychain, the VH5-3 variable heavy chain, the VH5-11 variable heavy chain,or the consensus variable heavy chain provided herein and referred to asthe consensus variable heavy chain region sequence 1 (consensus VHsequence 1). Exemplary variable heavy chain sequences for the hENT1antibodies of the invention also include the VH1-1 variable heavy chain,the VH1-4 variable heavy chain, the VH1-6 variable heavy chain, theVH4-2 variable heavy chain, the VH4-3 variable heavy chain, the VH4-4variable heavy chain or the consensus variable heavy chain providedherein and referred to as the consensus variable heavy chain regionsequence 2 (consensus VH sequence 2). The variable domain of each heavychain sequence is shown in bold in the sequences below.

Preferably, the variable heavy chains of the hENT1 antibodies of theinvention include a variable heavy chain complementarity determiningregion 1 (VH CDR1) sequence comprising the amino acid sequence GYTFTDYE(SEQ ID NO: 10), a variable heavy chain complementarity determiningregion 2 (VH CDR2) sequence comprising the amino acid sequence IDPETGAI(SEQ ID NO: 11) or the amino acid sequence IDPETGKT (SEQ ID NO: 40), anda variable heavy chain complementarity determining region 3 (VH CDR3)sequence comprising the amino acid sequence TREFTY (SEQ ID NO: 12) orthe amino acid sequence TRELTY (SEQ ID NO: 41). The CDRs were identifiedusing IMGT algorithms. (Lefranc, et al., Dev. Comp. Immunol., 27, 55-77(2003); Brochet et al., “IMGT/V-QUEST: the highly customized andintegrated system for IG and TR standardized V-J and V-D-J sequenceanalysis,” Nucl. Acids Res., vol. 36: W503-508 (2008)).

The VH3-12 heavy chain variable region (SEQ ID NO: 2) is encoded by thenucleic acid sequence shown in SEQ ID NO: 1. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 2. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 2.

>VH3-12 nucleic acid sequence (SEQ ID NO: 1)ATGGAATGCACCTGGGTTTTTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCCGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATTGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCCACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTTCCCCCTGGCACAAGCCAAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATCTATTCTTTGGGAA >VH3-12 amino acid sequence(SEQ ID NO: 2)

TVSAAKTTPPSVFPLAQAKFCRYPSHWRPLEHLFFG

The VH5-9 heavy chain variable region (SEQ ID NO:4) is encoded by thenucleic acid sequence shown in SEQ ID NO:3. The variable domain is shownin bold in the amino acid sequence shown in SEQ ID NO: 4. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 4.

>VH5-9 nucleic acid sequence  (SEQ ID NO: 3)ATGGAATGCACCTGGGTTATTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATCGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACAATCAGAAGCTCAAGGGCAAGGCCACACTGGCTGCAGACAAATCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTTTATCCACTGGCCCCCTGGAAGCTTGGG >VH5-9 amino acid sequence (SEQ ID NO: 4)

TVSAAKTTPPPVYPLAPWKLG

The VH5-12 heavy chain variable region (SEQ ID NO:6) is encoded by thenucleic acid sequence shown in SEQ ID NO:5. The variable domain is shownin bold in the amino acid sequence shown in SEQ ID NO: 6. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 6.

>VH5-12 nucleic acid sequence  (SEQ ID NO: 5)ATGAAATGGACCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATTGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTTTATCCACTGGCCCCTGGAAGCTTGGG >VH5-12 amino acid sequence (SEQ ID NO: 6)

TVSAAKTTPPSVYPLAPGSL

The VH5-13 heavy chain variable region (SEQ ID NO:8) is encoded by thenucleic acid sequence shown in SEQ ID NO:7. The variable domain is shownin bold in the amino acid sequence shown in SEQ ID NO: 8. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 8.

>VH5-13 nucleic acid sequence  (SEQ ID NO: 7)ATGGAATGCAGCAGGGTTATTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATTGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCCTGGCCCCTGGAAGCTTGGG >VH5-13 amino acid sequence (SEQ ID NO: 8)

TVSAAKTTPPPVYPLAPGSL

The VH5-3 heavy chain variable region (SEQ ID NO:51) is encoded by thenucleic acid sequence shown in SEQ ID NO:50. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 51. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 51.

>VH5-3 nucleic acid sequence  (SEQ ID NO: 50)ATGGAATGCACCTGGGTTCTTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATTGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACGATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCCTGGCCCCTGGAAGCTTGGG >VH5-3 amino acid sequence (SEQ ID NO: 51)

TVSAAKTTPPPVYPLAPGSL

The VH5-11 heavy chain variable region (SEQ ID NO:53) is encoded by thenucleic acid sequence shown in SEQ ID NO:52. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 53. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 53.

>VH5-11 nucleic acid sequence  (SEQ ID NO: 52)ATGGGATGGAGCGTGGTTTATCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCATCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGCCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAACAGACACCTGTGCATGGCCTGGAATGGATTGGCGCTATTGATCCTGAAACTGGTGCTATTGTCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTTTATCCCCTGGTCCCTGGAAGCTTGGG >VH5-11 amino acid sequence (SEQ ID NO: 53)

TVSAAKTTPPSVYPLVPGSL

The amino acid sequence of the consensus heavy chain variable regionsequence 1 is shown in SEQ ID NO: 9. The variable domain is shown inbold in the amino acid sequence shown in SEQ ID NO: 9. The CDR regionsare boxed in the amino acid sequence shown in SEQ ID NO: 9.

>Consensus VH amino acid sequence 1  (SEQ ID NO: 9)

TVSAAKTTPPSVYPLAPGSL

An alignment of these variable heavy chain sequences is shown below:

1 50 VH5-13 (1) MECSRVILFLLSVIAGVQSQVHLQQSGAELVRPGASVTLPCKASGYTFTD VH5-9(1) MECTWVILFLLSVIAGVQSQVHLQQSGAELVRPGASVTLPCKASGYTFTD VH5-12 (1)MKWTWVFLFLLSVIAGVQSQVHLQQSGAELVRPGASVTLPCKASGYTFTD VH3-12 (1)MECTWVFLFLLSVIAGVQSQVHLQQSGAELVRPGASVTPPCKASGYTFTD VH5-3 (1)

VH5-11 (1)

.****.*..............................*............ Consensus 1 (1)MECTWVILFLLSVIAGVQSQVHLQQSGAELVRPGASVTLPCKASGYTFTD 51 100 VH5-13 (51)

VH5-9 (51)

VH5-12 (51)

VH3-12 (51)

VH5-3 (51)

VH5-11 (51)

...............................*.....*...........*. Consensus 1 (51)YEMHWVKQTPVHGLEWIGAIDPETGAIVYNQKFKGKATLTADKSSNTAYM 101 150 VH5-13 (101)ELRSLTSEDSAVYYCTREFTYWGQGTLVTVSAAKTTPPPVYPLAPGSL-- VH5-9 (101)

VH5-12 (101)

VH3-12 (101)

VH5-3 (101)

VH5-11 (101)

...........................................*.*...**** Consensus 1 (101)ELRSLTSEDSAVYYCTREFTYWGQGTLVTVSAAKTTPPSVYPLAPGSL 151 164 VH5-13 (149)---------------(SEQ ID NO: 8) VH5-9 (150) ---------------(SEQ ID NO: 4)VH5-12 (149) ---------------(SEQ ID NO: 6) VH3-12 (151)YPSHWRPLEHLFFG (SEQ ID NO: 2) VH5-3 (149) ---------------(SEQ ID NO: 51)VH5-11 (149) ---------------(SEQ ID NO: 53) Consensus 1 (151)                (SEQ ID NO: 9)

The VH1-1 heavy chain variable region (SEQ ID NO: 28) is encoded by thenucleic acid sequence shown in SEQ ID NO: 27. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 28. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 28.

>VH1-1 nucleic acid sequence  (SEQ ID NO: 27)ATGAAATGCAGCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCGGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGTACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCGGTCTTCCCCCTGGCAC >VH1-1 amino acid sequence (SEQ ID NO: 28)

TVSAAKTTPPSVFPLA

The VH1-4 heavy chain variable region (SEQ ID NO: 30) is encoded by thenucleic acid sequence shown in SEQ ID NO: 29. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 30. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 30.

>VH1-4 nucleic acid sequence  (SEQ ID NO: 29)ATGGAATGCACCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGGAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGTACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTCTTCCCCCTGGCAC >VH1-4 amino acid sequence (SEQ ID NO: 30)

TVSAAKTTPPSVFPLA

The VH1-6 heavy chain variable region (SEQ ID NO: 32) is encoded by thenucleic acid sequence shown in SEQ ID NO: 31. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 32. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 32.

>VH1-6 nucleic acid sequence  (SEQ ID NO: 31)ATGAAATGCAGCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCCCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGTACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCGGTCTTCCCCCTGGCAC >VH1-6 amino acid sequence (SEQ ID NO: 32)

TVSAAKTTPPSVFPLA

The VH4-2 heavy chain variable region (SEQ ID NO: 34) is encoded by thenucleic acid sequence shown in SEQ ID NO: 33. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 34. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 34.

>VH4-2 nucleic acid sequence  (SEQ ID NO: 33)ATGAAATGCAGCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGCACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCATTGGCCCCTGGAAGCTTGGG >VH4-2 amino acid sequence (SEQ ID NO: 34)

TVSAAKTTPPPVYPLAPGSL

The VH4-3 heavy chain variable region (SEQ ID NO: 36) is encoded by thenucleic acid sequence shown in SEQ ID NO: 35. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 36. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 36.

>VH4-3 nucleic acid sequence  (SEQ ID NO: 35)ATGAAATGGACCTGGGTTTTTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGTACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCCCTGGCCCCTGGAAGCTTGGG >VH4-3 amino acid sequence (SEQ ID NO: 36)

TVSAAKTTPPPVYPLAPGSL

The VH4-4 heavy chain variable region (SEQ ID NO: 38) is encoded by thenucleic acid sequence shown in SEQ ID NO: 37. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 38. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 38.

>VH4-4 nucleic acid sequence  (SEQ ID NO: 37)ATGGAATGGAGCTGGGTTTTCCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGTCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATAGAGCGATTGATCCTGAAACTGGTAAAACTGCCTACAATCAGAAGTTCAAGGGCAAGACCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGTTCCGCAGCCTGACATCTGAGGACTCTGCCGTCCATTACTGTACAAGAGAGTTGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCACCCGTCTATCCATTGGCCCCCTGGAAGCTTGGG >VH4-4 amino acid sequence (SEQ ID NO: 38)

TVSAAKTTPPPVYPLAPWKLG

The amino acid sequence of the consensus heavy chain variable regionsequence 2 is shown in SEQ ID NO: 39. The variable domain is shown inbold in the amino acid sequence shown in SEQ ID NO: 39. The CDR regionsare boxed in the amino acid sequence shown in SEQ ID NO: 39.

>Consensus VH amino acid sequence 2  (SEQ ID NO: 39)

TVSAAKTTPPSVFPLAP

An alignment of these variable heavy chain sequences is shown below:

1                                               50 VH1-1   (1)MKCSWVFLFLLSVIAGVQSRVQLQQSGSELVRPGASVTLSCKASGYTFTD VH1-6   (1)MKCSWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD VH1-4   (1)MECTWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD VH4-2   (1)MKCSWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD VH4-3   (1)MKWTWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD VH4-4   (1)MEWSWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD Consensus 2   (1)MKCSWVFLFLLSVIAGVQSQVQLQQSGSELVRPGASVTLSCKASGYTFTD51                                             100 VH1-1   (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM VH1-6 (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSPSTAYM VH1-4 (51)YEMHWVEQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM VH4-2 (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM VH4-3 (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM VH4-4 (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM Consensus 2 (51)YEMHWVKQTPVHGLEWIGAIDPETGKTAYNQKFKGKTTLTADKSSSTAYM101                                            149 (SEQ NO) VH1-1 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPSVFPLA----- (28) VH1-6 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPSVFPLA----- (32) VH1-4 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPSVFPLA----- (30) VH4-2 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPPVYPLAPGSL- (34) VH4-3 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPPVYPLAPGSL- (36) VH4-4 (101)EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPPVYPLAPWKLG  (38) Consensus 2(101) EFRSLTSEDSAVHYCTRELTYWGQGTLVTVSAAKTTPPSVFPLAP  L (39)

Exemplary variable light chain sequences for the hENT1 antibodies of theinvention include the VL2 variable light chain, the VL10 variable lightchain, the VL11 variable light chain, the VL20 variable light chain, theVL21 light chain, or the consensus variable light chain provided hereinand referred to as the consensus variable light chain region sequence 1(consensus VL sequence 1). Exemplary variable light chain sequences forthe hENT1 antibodies of the invention also include the VL2-2 variablelight chain, the VL2-3 variable light chain, the VL2-7 variable lightchain, the VL2-10 variable light chain, the VL2-12 variable light chain,the VL2-16 variable light chain or the consensus variable light chainprovided herein and referred to as the consensus variable light chainregion sequence 2 (consensus VL sequence 2).

Preferably, the variable light chains of the hENT1 antibodies of theinvention include a variable light chain complementarity determiningregion 1 (VL CDR1) sequence comprising the amino acid sequenceQSLLFSNGKTY (SEQ ID NO: 24), a variable light chain complementaritydetermining region 2 (VL CDR2) sequence comprising the amino acidsequence LVS (SEQ ID NO: 25), and a variable light chain complementaritydetermining region 3 (VL CDR3) sequence comprising the amino acidsequence VQGTHFPWT (SEQ ID NO: 26). The CDRs were identified using IMGTalgorithms. (Lefranc, et al., Dev. Comp. Immunol., 27, 55-77 (2003);Brochet et al., “IMGT/V-QUEST: the highly customized and integratedsystem for IG and TR standardized V-J and V-D-J sequence analysis,”Nucl. Acids Res., vol. 36: W503-508 (2008)).

The VL2 light chain variable region (SEQ ID NO: 14) is encoded by thenucleic acid sequence shown in SEQ ID NO: 13. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 14. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 14.

>VL2 nucleic acid sequence  (SEQ ID NO: 13)TCTCCTTTCAACAAAGCACATTTCGATTTCCAGCTTGGTGCCTCCACCGAACGTCCACGGAAAATGTGTACCTTGCACGCAGTAATAAACTCCCAAATCCTCAGCCTCCACTCTGCTGATTTTCAGTGAAAAATCTGTTCCTGAACCAGTGCCAGTGAACCTGTCAGGGACTCCAGAGTTCAGTTTAGACACCAGATAGATTAGGCGCTTTGGAGACTGGCCTGGCCTCTGAAATAACCAATTCAAGTAGGTTTTTCCATTACTAAATAAGAGGCTCTGACTTGACCTGCAAGAGACAGAGGCTGGTTGTCCAATGGTAACCGACAAAGTGAGTGGAGTTTGGGTCATCAAAACATC >VL2 amino acid sequence (SEQ ID NO: 14)

The VL10 light chain variable region (SEQ ID NO: 16) is encoded by thenucleic acid sequence shown in SEQ ID NO: 15. The CDR regions are boxedin the amino acid sequence shown in SEQ ID NO: 16.

>VL10 nucleic acid sequence (SEQ ID NO: 15)ATTGGATATCCTCGCAGCATCTCGGCTTGATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTGTCTCTTGCAGGTCAAGTCAGAGCCTCTTATTTAGTAATGGAAAAACCTATTTGAATTGGTTATTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGAACTCTGGAGTCCCTGACAGGTTCACTGGCACTGGTTCAGGAACAGATTTTTCACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG >VL10 amino acid sequence (SEQ ID NO: 16)

The VL11 light chain variable region (SEQ ID NO: 18) is encoded by thenucleic acid sequence shown in SEQ ID NO: 17. The CDR regions are boxedin the amino acid sequence shown in SEQ ID NO: 18.

>VL11 nucleic acid sequence (SEQ ID NO: 17)CTTTCGCGATGATCCTTGCACGCATTTCAGGCTTGGATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTGTCTCTTGCAGGTCAAGTCAGAGCCTCTTATTTAGTAATGGAAAAACCTATTTGAATTGGTTATTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTGTCTGGTGTCTAAACTGAACTCTGGAGTCCCTGACAGGTTCACTGGCACTGGTTCAGGAACAGATTTTTCACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGATTTTATTACTGCGTGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG >VL11 amino acid sequence(SEQ ID NO: 18)

The VL20 light chain variable region (SEQ ID NO: 20) is encoded by thenucleic acid sequence shown in SEQ ID NO: 19. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 20. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 20.

>VL20 nucleic acid sequence (SEQ ID NO: 19)GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTGTCTCTTGCAGGTCAAGTCAGAGCCTCTTATTTAGTAATGGAAAAAACCTATTTGAATTGGTTATTTCAGAGGCCAGGCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGAACTCTGGAGTCTCTGACAGGTTCACTGGCACTGGTTCAGAAACAGATTTTTCACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACATATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGCCCTTTTTAATTCTGCAGATATCCTATCACAACGTTGCTGGCCGCGGCCGCT >VL20 amino acid sequence(SEQ ID NO: 20)

The VL21 light chain variable region (SEQ ID NO: 22) is encoded by thenucleic acid sequence shown in SEQ ID NO: 21. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 22. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 21.

>VL21 nucleic acid sequence (SEQ ID NO: 21)GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTGTCTCTTGCAGGTCAAGTCAGAGCCTCTTATTTAGTAATGGAAAAACCTATTTGAATTGGTTATTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGAACTCTGGAGTCCCTGACAGGTTCACCGGCACTGGTTCAGGAACAGATTTTCCACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCGTGGACGTTCGGTGCCCTTTTTAAAGGAGGCCGTGATAAAAAAT >VL21 amino acid sequence(SEQ ID NO: 22)

The consensus light chain variable region sequence 1 is encoded by thenucleic acid sequence shown in SEQ ID NO: 23. The CDR regions are boxedin the amino acid sequence shown in SEQ ID NO: 23.

>Consensus VL amino acid sequence 1 (SEQ ID NO: 23)

An alignment of these variable light chain sequences is shown below:

The VL2-2 light chain variable region (SEQ ID NO: 43) is encoded by thenucleic acid sequence shown in SEQ ID NO: 42. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 43. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 43.

>VL2-2 nucleic acid sequence (SEQ ID NO: 42)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCTGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCCGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCTCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAAAG >VL2-2 amino acid sequence(SEQ ID NO: 43)

The VL2-3 light chain variable region (SEQ ID NO: 45) is encoded by thenucleic acid sequence shown in SEQ ID NO: 44. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 45. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 45.

>VL2-3 nucleic acid sequence (SEQ ID NO: 44)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCTGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCTGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCTCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAGAGA >VL2-3 amino acid sequence(SEQ ID NO: 45)

The VL2-7 light chain variable region (SEQ ID NO: 45) is encoded by thenucleic acid sequence shown in SEQ ID NO: 44. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 45. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 45.

>VL2-7 nucleic acid sequence (SEQ ID NO: 44)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCTGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCTGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCATCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAGAGA >VL2-7 amino acid sequence (SEQ ID NO: 45)

The VL2-10 light chain variable region (SEQ ID NO: 47) is encoded by thenucleic acid sequence shown in SEQ ID NO: 46. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 47. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 47.

>VL2-10 nucleic acid sequence (SEQ ID NO: 46)ATGAAGTTGCCTGTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCAGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCTGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCTCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAGAGA >VL2-10 amino acid sequence(SEQ ID NO: 47)

The VL2-12 light chain variable region (SEQ ID NO: 43) is encoded by thenucleic acid sequence shown in SEQ ID NO: 48. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 43. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 43.

>VL2-12 nucleic acid sequence (SEQ ID NO: 48)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCTGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCTGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCTCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAAAGA >VL2-12 amino acid sequence (SEQ ID NO: 43)

The VL2-16 light chain variable region (SEQ ID NO: 45) is encoded by thenucleic acid sequence shown in SEQ ID NO: 44. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 45. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 45.

>VL2-16 nucleic acid sequence (SEQ ID NO: 44)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCCGCCTCCTCCTCCGACGTGCTGATGACCCAGACCCCCCTGACCCTGTCCGTGACCATCGGCCAGCCTGCCTCTGTGTCCTGCCGGTCCTCCCAGTCCCTGCTGTTCTCCAACGGCAAGACCTACCTGAACTGGCTGTTCCAGCGGCCTGGCCAGTCCCCCCAAGCGGCTGATCTACCTGGTGTCCAAGCTGAACTCCGGCGTGCCCGACCGGTTTACAGGCACCGGCTCTGGCACCGACTTCAGCCTGAAGATCAGCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGCGTGCAGGGCACCCACTTCCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCCGATGCCGCCCCTACCGTGTCCATCTTCCCACCCTCCAGCGAGCAGCTGACCTCTGGCGGCGCTTCCGTCGTGTGCTTCCTGAACAACTTCTACCCCAGAGA >VL2-16 amino acid sequence (SEQ ID NO: 45)

The consensus light chain variable region sequence 2 is encoded by thenucleic acid sequence shown in SEQ ID NO: 49. The variable domain isshown in bold in the amino acid sequence shown in SEQ ID NO: 49. The CDRregions are boxed in the amino acid sequence shown in SEQ ID NO: 49.

>Consensus VL amino acid sequence 2 (SEQ ID NO: 49)

An alignment of these variable light chain sequences is shown below:

1                                               50 VL2-16 (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF VL2-12 (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF VL2-2 (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF VL2-7 (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF VL2-10 (1)-EVACRLLVLMFWIPASSSDVQMTQTPLTLSVTIGQPASVSCRSSQSLLF VL2-3 (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF Consensus (1)MKLPVRLLVLMFWIPASSSDVLMTQTPLTLSVTIGQPASVSCRSSQSLLF51                                             100 VL2-16 (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS VL2-12 (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS VL2-2 (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS VL2-7 (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS VL2-10 (50)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS VL2-3 (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS Consensus (51)SNGKTYLNWLFQRPGQSPKRLIYLVSKLNSGVPDRFTGTGSGTDFSLKIS101                                            150 VL2-16 (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT VL2-12 (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT VL2-2 (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT VL2-7 (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT VL2-10 (100)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT VL2-3 (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT Consensus (101)RVEAEDLGVYYCVQGTHFPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLT 151          166VL2-16 (151) SGGASVVCFLNNFYPR (SEQ ID NO: 45) VL2-12 (151)SGGASVVCFLNNFYPK (SEQ ID NO: 43) VL2-2 (151)SGGASVVCFLNNFYPK (SEQ ID NO: 43) VL2-7 (151)SGGASVVCFLNNFYPR (SEQ ID NO: 45) VL2-10 (150)SGGASVVCFLNNFYPR (SEQ ID NO: 47) VL2-3 (151)SGGASVVCFLNNFYPR (SEQ ID NO: 45) Consensus (151)SGGASVVCFLNNFYPR (SEQ ID NO: 49)

Also included in the invention are antibodies that bind to the sameepitope as the antibodies described herein. For example, antibodies ofthe invention specifically bind to hENT1, wherein the antibody binds toan epitope that includes one or more amino acid residues on human hENT1(see e.g., Accession Nos. AAC51103.1; NP_(—)001071645.1;NP_(—)001071644.1; NP_(—)0010171643.1; NP_(—)001071642.1;NP_(—)004946.1; NP_(—)001523.2; AAM11785.1; AAF02777.1).

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody hasthe same specificity as a monoclonal antibody of the invention byascertaining whether the former prevents the latter from binding tohENT1. If the monoclonal antibody being tested competes with themonoclonal antibody of the invention, as shown by a decrease in bindingby the monoclonal antibody of the invention, then the two monoclonalantibodies bind to the same, or a closely related, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with soluble hENT1protein and then add the monoclonal antibody being tested to determineif the monoclonal antibody being tested is inhibited in its ability tobind hENT1. If the monoclonal antibody being tested is inhibited then,in all likelihood, it has the same, or functionally equivalent, epitopicspecificity as the monoclonal antibody of the invention. Screening ofmonoclonal antibodies of the invention, can be also carried out, e.g.,by measuring and determining whether the test monoclonal antibody isable to modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with hENT1 expression and/or activity.

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against hENT1, or against derivatives,fragments, analogs homologs or orthologs thereof (See, e.g., Antibodies:A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., incorporated herein byreference). Fully human antibodies are antibody molecules in which theentire sequence of both the light chain and the heavy chain, includingthe CDRs, arise from human genes. Such antibodies are termed “humanantibodies,” or “fully human antibodies” herein. Human monoclonalantibodies are prepared, for example, by using the trioma technique; thehuman B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today4: 72); and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies maybe utilized and may be produced by using human hybridomas (see Cote, etal., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforminghuman B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The antibodies of the invention are monoclonal antibodies. Monoclonalantibodies that modulate, block, inhibit, reduce, antagonize, neutralizeor otherwise interfere with hENT1 expression and/or activity aregenerated, e.g., by immunizing an animal with hENT1 such as, forexample, murine, rat or human hENT1 or an immunogenic fragment,derivative or variant thereof. Alternatively, the animal is immunizedwith cells transfected with a vector containing a nucleic acid moleculeencoding hENT1, such that hENT1 is expressed and associated with thesurface of the transfected cells. Alternatively, the antibodies areobtained by screening a library that contains antibody or antigenbinding domain sequences for binding to hENT1. This library is prepared,e.g., in bacteriophage as protein or peptide fusions to a bacteriophagecoat protein that is expressed on the surface of assembled phageparticles and the encoding DNA sequences contained within the phageparticles (i.e., “phage displayed library”). Hybridomas resulting frommyeloma/B cell fusions are then screened for reactivity to hENT1.

Monoclonal antibodies of the invention include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

In some methods, a hENT1 antibody is developed, for example, usingphage-display methods using antibodies containing only human sequences.Such approaches are well-known in the art, e.g., in WO92/01047 and U.S.Pat. No. 6,521,404, which are hereby incorporated by reference. (Seealso Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPlückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743).

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and an immune variable region,it is expected that certain human anti-chimeric antibody (HACA)responses will be observed, particularly in chronic or multi-doseutilizations of the antibody. Thus, it would be desirable to providefully human antibodies against hENT1 in order to vitiate or otherwisemitigate concerns and/or effects of HAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92/102190 and U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG3 and IgG4. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)anti-hENT1 fragments, single chain anti-hENT1 antibodies, bispecificanti-hENT1 antibodies, and heteroconjugate anti-hENT1 antibodies.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberranthENT1 expression and/or activity. For example, cysteine residue(s) canbe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Those ofordinary skill in the art will recognize that a large variety ofpossible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines,” Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference). Coupling may be accomplished by anychemical reaction that will bind the two molecules so long as theantibody and the other moiety retain their respective activities. Thislinkage can include many chemical mechanisms, for instance covalentbinding, affinity binding, intercalation, coordinate binding andcomplexation. The preferred binding is, however, covalent binding.

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

Use of Antibodies Against hENT1

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablepharmaceutically acceptable carriers, excipients, and other agents thatare incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences (15th ed, Mack PublishingCompany, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour,therein. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as Lipofectin™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax.

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used.

Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. A pharmaceutical compositionof the invention is formulated to be compatible with its intended routeof administration. Examples of routes of administration includeparenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,inhalation), transdermal (i.e., topical), transmucosal, and rectaladministration. The pharmaceutical compositions can be included in acontainer, pack, or dispenser together with instructions foradministration.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. The amount required to be administered will depend on thebinding affinity of the antibody for its specific antigen, and will alsodepend on the rate at which an administered antibody is depleted fromthe free volume other subject to which it is administered. Common rangesfor therapeutically effective dosing of an antibody or antibody fragmentof the invention may be, by way of nonlimiting example, from about 0.1mg/kg body weight to about 50 mg/kg body weight. Common dosingfrequencies may range, for example, from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular cancer or neoplasticdisorder. Alleviation of one or more symptoms of the cancer orneoplastic disorder indicates that the antibody confers a clinicalbenefit.

In another embodiment, antibodies directed against hENT1 may be used inmethods known within the art relating to the localization and/orquantitation of hENT1 (e.g., for use in measuring levels of the hENT1within appropriate physiological samples, for use in diagnostic methods,for use in imaging the protein, and the like). In yet anotherembodiment, an antibody according to the invention can be used as anagent for detecting the presence of hENT1 (or a protein fragmentthereof) in a sample. In another embodiment, an antibody specific forhENT1 can be used to isolate a hENT1 polypeptide by standard techniques,such as immunoaffinity, chromatography or immunoprecipitation. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes (e.g., horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase), prosthetic groups (e.g.,streptavidin/biotin and avidin/biotin), fluorescent materials (e.g.,umbelliferone, fluorescein, fluorescein isothiocyanate), luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin), and radioactive materials (e.g., ¹²⁵I, ¹³¹I, ³⁵S or ³H).

Diagnostic and Prophylactic Formulations

hENT1 monoclonal antibodies of the invention are used in diagnostic andprophylactic formulations. In one embodiment, a hENT1 antibody is usedto identify patients or patient samples where hENT1 expression and/oractivity is low or otherwise reduced. In one embodiment, a hENT1antibody of the invention is used to identify patients that are morelikely to have successful treatment of a cancer or other neoplasticdisorder based on the detected hENT1 expression and/or activity level.In one embodiment, a hENT1 antibody of the invention is used to identifypatients that are at risk of developing a disorder associated withaberrant hENT1 expression and/or activity. In one embodiment, a hENT1antibody of the invention is administered to patients that are at riskof developing a disorder associated with aberrant hENT1 expressionand/or activity. A patient's or organ's predisposition to one or more ofthese disorders can be determined using genotypic, serological orbiochemical markers such as, for example, hENT1 expression and/oractivity level.

Antibodies of the invention are also useful in the detection of hENT1 inpatient samples and accordingly are useful as diagnostics. For example,the hENT1 antibodies of the invention are used in in vitro assays, e.g.,ELISA, to detect hENT1 levels in a patient sample.

In one embodiment, a hENT1 antibody of the invention is immobilized on asolid support (e.g., the well(s) of a microtiter plate). The immobilizedantibody serves as a capture antibody for any hENT1 that may be presentin a test sample. The level of detectable label is measured, and theconcentration of hENT1 antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Anti-hENT1 Antibody Generation and Sequencing

Antibodies were generated as described in Jennings et al., “Distinctregional distribution of human equilibrative nucleoside transporterproteins 1 and 2 (hENT1 and hENT2) in the central nervous system,”Neuropharmacology, vol. 40(5): 722-731 (2001), the contents of which arehereby incorporated by reference in their entirety. Briefly, monoclonalantibodies specific for the hENT1 protein were produced by immunizationof mice with a synthetic peptide that corresponded to amino acids254-271 of the predicted intracellular loop between transmembranesegments 6 and 7 of hENT1 and was conjugated to keyhole limpethemocyanin (KLH). The synthetic peptide-KLH conjugate, purified to >95%homogeneity, was from the Alberta Peptide Institute (University ofAlberta, Canada). Monoclonal antibody production followed the proceduresof Harlow and Lane (“Antibodies: a Laboratory Manual,” Harlow and Lane(eds.) (1988)). Hybridomas were: (i) produced by fusion of splenocyteswith the murine myeloma non-secreting cell line, PC/NSI/1-AG4-1; (ii)selected by their ability to grow in 100 μM hypoxanthine, 16 μMaminopterin and 0.4 μM thymidine; and (iii) maintained in growth mediumthat consisted of RPMI 1640 supplemented with 20% heat-inactivated fetalbovine serum. The supernatants from hybridoma cultures were screened forimmunoreactivity by enzyme-linked immunoabsorbent assay (ELISA) usingthe synthetic peptide (hENT1 amino acids 254-271) conjugated to bovineserum albumin. Hybridoma cultures that exhibited a strong positiveresult on ELISA in three separate assays were subjected to limitingdilution to obtain clonal populations. Supernatants from the hybridomacultures that produced monoclonal antibodies with high specificity andavidity for hENT1 protein were collected, centrifuged at 1000 g for 10minutes to remove any cellular debris and stored at −20° C. until use.

mRNA was extracted from the hybridoma cell pellets, and total RNA wasextracted from the pellets. RT-PCR was performed on the extracted RNA.Briefly, cDNA was created from the RNA by reverse-transcription with anoligo(dT) primer. PCR reactions using variable domain primers to amplifyboth the VH and VL regions of the monoclonal antibody DNA. The VH and VLbands were isolated, and the VL band was gel purified. Both the VL andVH PCR products were cloned into the Invitrogen sequencing vector pCR2.1(Invitrogen by Life Technologies, Carlsbad Calif.) and transformed intoTOP10 E. coli cells (Invitrogen by Life Technologies, Carlsbad Calif.)for positive transformants. Selected colonies, VH3-12, VH5-9, VH5-12,VH5-13, VH1-1, VH1-4, VH1-6, VH4-2, VH4-3, VH4-4, VL2, VL10, VL11, VL20,VL21, VL2-2, VL2-3, VL2-7, VL2-10, VL2-12 and VL2-16 were picked andanalyzed through sequencing. These VH and VL sequences are describedabove.

Antibodies were tested for binding and functional activity using theassays described herein. Briefly, the expression of antibody isqualified and quantified using sandwich ELISA. A calibration standard isestablished using mouse Sigma IgG1 Kappa (Sigma-Aldrich, St. Louis,Mo.). The absorbance is measured at wavelength 450 nM, and the amount ofmouse IgG1 Kappa is determined. A second ELISA is then used todemonstrate the affinity of the hENT1 antibody to the hENT1 polypeptideof amino acids 254-271 conjugated to bovine serum albumin. A secondaryantibody (goat anti mouse IgG conjugated to horse radish peroxidase) isused and the ELISA plates are read at 450 nM.

Example 2 Expression of Anti-hENT1 Antibodies

The studies described herein demonstrate the expression of anti-hENT1IgG₁ antibodies produced by transfection of CHO cells.

In the first study, following heavy and light chain amino acid sequenceswere synthesized and cloned into the antibody expression plasmid shownin FIG. 1. The DNA coding for the antibody light chain and heavy chainabove was synthesized and optimized for expression in CHO cells byGeneart (Germany). The DNA was inserted into a DHFR expression vector.This vector can also be used for the development of a stable cell line,by selection with Neomycin/G418 antibiotic and/or methotrexate (a DHFRinhibitor).

Heavy chain amino acid sequence (SEQ ID NO: 54)

LYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVL HEGLHNHHTEKSLSHSPGKLight chain amino acid sequence (SEQ ID NO: 55)

KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The variable domains of the heavy and light chains are shown in bold inSEQ ID NO: 54 (heavy chain) and SEQ ID NO: 55 (light chain). Thecomplementarity determining regions (CDRs) are shown in boxes in SEQ IDNO: 54 (heavy chain) and SEQ ID NO: 55 (light chain). CDR sequences wereidentified using IMGT algorithms as described herein.

Several transfections were carried out on the antibody expressionplasmid, under varying conditions in order to optimize the transfectionefficiency. Transient transfection (CHO method) was performed asfollows: (1) CHO-S cells were cultured in PROCHO4 CDM medium (Lonza);(2) On the day of transfection the cells were centrifuged at 200 g for 5min and resuspended in fresh UltraCHO/PROCHO5 CDM medium (Lonza) at acell density of 2×10⁶ cells/ml; (3) Transfection of culture wasperformed in a spinner flask using 2.5 μg of DNA and either 4 to 1 or 6to 1 of PEI to DNA ratio per 1 ml of culture, diluted in 150 mM NaCl;(4) After 5 h, the transfected culture were diluted with 500 ml of freshUltraCHO/ProCHO5 medium and incubated at 37° C. in 6% CO₂ with agitationat 88 rpm; and (5) The supernatant was collected after 7-12 dayspost-transfection.

The following additional transient transfections were also run:

Transient Transfection with PEI 4:1 Ratio:

The transfection was carried out in UltraCHO medium at a volume size of600 ml in a spinner flask. A DNA plasmid prep at a concentration of 1mg/ml was used. Supernatant was harvested eight days later forpurification (“Supernatant 1”).

Transient Transfection with PEI 4:1 Ratio:

Transfection was repeated with UltraCHO medium at a volume size 932 mlin spinner flask. The same plasmid prep was used as above. Supernatantwas harvested eight days later for purification.

Transient Transfection with PEI 4:1 Ratio:

Transfection was carried out in 700 ml UltraCHO medium with freshMaxiprep at a concentration of 2470 μg/ml and purity of 1.7. Supernatantwas collected and frozen 11 days later for further purification(“Supernatant 2”)

Transient Transfection with PEI 4:1 Ratio:

Transfection was repeated with the same Maxiprep used above in 1000 mlUltraCHO medium. Supernatant was harvested seven days later forpurification (“Supernatant 3”).

Transient Transfection with PEI 6:1 Ratio:

Modifications were made on the PEI and DNA ratio, which changed from 4:1to 6:1 ratio, and ProCHO5 medium was used along with UltraCHO medium tocompare expression. Concentration of the plasmid prep was 1770 μg/ml andpurity was 1.7. Transfections were carried out in volume 700 ml UltraCHOand 500 ml ProCHO5 in spinner flasks. Supernatants were harvested sevendays later for purification.

Transient Transfection with PEI 6:1 Ratio:

Transfection was carried out on 480 ml ProCHO5 medium in three separateErlenmeyer flasks. The concentration of plasmid prep was 1135 μg/ml andpurity was 1.61. Supernatant was harvested several days later.

Transient Transfection with PEI 6:1 Ratio:

Transfection was carried out on 200 ml ProCHO5 medium in an Erlenmeyerflask. The concentration of plasmid prep was 370 m/ml and purity was1.5.

Transient Transfection with PEI 6 to 1 Ratio:

Transfection volume was 500 ml in ProCHO5 medium on Erlenmeyer flasks.The concentration of plasmid prep was 3645 m/ml and purity was 1.7.

The expressed antibody has been detected in the media, and the expressedantibody has been confirmed as anti-hENT1 by ELISA.

The anti-hENT1 antibody was purified from 2 batches of cell culturesupernatant of 200 ml and 500 ml using IgG binding buffers. Thepurifications successfully yielded anti-hENT1 antibody, as demonstratedby Western blot. However, there was also a protein band around 100 kDain the purified sample.

Supernatants from several transfections described above were tested byquantitative ELISA along with murine IgG positive control (Sigma-IgGfrom murine serum). The tested supernatants are referred to herein as“Supernatant 1,” “Supernatant 2,” and “Supernatant 3.” The ELISA plate(Nunc Maxisorp) was coated with goat anti mouse IgG antibodies (Sigma)at concentration 100 ng/well overnight at 4° C. The plate was thenblotted with 3% milk (Marvel dried skimmed milk powder) for two hours atroom temperature with 150 rpm shaking Murine IgG was prepared in serialdilution from 10 ng/well to 0 ng/well, and added in triplicate to theplate as standard curve. 100 μl of supernatant samples was added intriplicate on the plate. The plate was incubated at room temperaturewith 150 rpm shaking for two hours and washed with PBS-0.1% Tween 6times. Anti-mouse IgG antibody (Bio-Rad, Goat Anti-Mouse IgG (H+L)-HRPconjugate) was added for one hour with 150 rpm shaking, then washed withPBS-0.1% Tween 6 times and once with PBS before being developed withTMB. The results of this quantitative ELISA are shown in FIG. 2.

All three supernatants were then tested using a non-reducing westernblot methodology. The non-reducing gel was prepared with 12% acrylamidewithout SDS, and 30 μl of each supernatant samples (Sup. 1, Sup. 2 andSup. 3) were mixed with treatment buffer at 5:1 dilution respectively(Treatment buffer contained stacking gel buffer, glycerol and doubledistilled water, SDS or β-mercaptoethanol were removed). Samples mixturewere loaded at 30 μl per well. The marker in Lane 1 is See Blue Plus 2Prestained Standard (1×) (Invitrogen). The gel was then run at 180V for85 minutes, and transferred to western blot membrane (AmershamBiosciences, Hybond-C Extra) at 20V for 25 minutes. The membrane wasblocked with 3% milk (Marvel dried skimmed milk powder) for two hours atroom temperature with shaking and probed with anti-mouse IgG antibody(Bio-Rad, Goat Anti-Mouse IgG (H+L)-HRP conjugate) overnight at 4° C.with shaking at 70 rpm. The blot was washed intensively with PBS-0.1%Tween and PBS the next day before developing with ECL solution (ThermoScientific, SuperSignal West Pico Chemiluminescent substrate) for 1 to 5mins.

As shown in FIG. 3, a band was visible between 148 kDa and 250 kDa bandsin Supernatant 2 and at the same size in the positive control murine IgG(FIG. 3). Due to the saturation of murine IgG control, the western blotwas developed again without the murine IgG (FIG. 4).

Bands are visible in both Supernatant 2 and Supernatant 3. The band inthe Supernatant 2 sample appeared to be strongest after beingover-developed, and a weak band was visible in the Supernatant 3 sample.This correlated with the concentrations determined by quantitative ELISAin FIG. 2. Both sample bands came up in the unconcentrated supernatantsat the same size as the band in the murine IgG positive control.

Intensity of the supernatant bands on the non-reducing western blotcorresponds to the concentration showed on quantitative ELISA.Supernatant 2 sample had the highest concentration of IgG and showed thestrongest band on western blot, Supernatant 3, which contained less IgG,showed a weak band on blot; and Supernatant 1 contained the lowestconcentration of IgG showed no band at all. The variation among thesesupernatants could be due to the transfection efficiency and IgGexpression. Supernatant samples 2 and 3 were transfected with the sameDNA preparation which could explain the relatively higher expressionlevels.

Bands were visible in Supernatant 2 and 3 samples 10 at the same size asthe positive control murine IgG by non-reducing western blot. Intactrecombinant murine IgG has been produced by transient transfection inCHO cells at a maximal concentration of 466 μg/L.

The isotype of the expressed, recombinant anti-hENT1 antibody wasdetermined using BioAssay Works lateral flow Mouse Iso-Gold test kits(KSOT-001). Briefly 5 μl of antibody samples from two different batcheswere added to 0.5 ml of sample diluent (ISOT-003) according to themanufacturer's instructions for the testing of “Cell culture/supernatantfluid”; 150 μl of the diluted samples were then added to the threelateral flow cartridges (ISOT-005) and the results were read at 5minutes. There was a weak result, indicative of low antibodyconcentration, for both samples for mouse IgG₁ with a kappa light chain,the correct isotype to mimic the product of the 10D7G2 hybridoma. Nocontaminant isoforms were observed.

Expression of the murine IgG₁ anti-hENT1 antibody was demonstrated usinga sandwich ELISA. Carbonate bicarbonate coating buffer was preparedaccording to the manufacturer's prescription (1 tablet per 100 ml) usingFluka carbonate bicarbonate buffer tablets. 100 μl of goat anti mouseIgG antibody (Bethyl) was diluted with 10 ml of the pH 9.6 coatingbuffer. A Greiner 96 well medium bind ELISA plate was coated at 100 μlper well with the anti mouse IgG plate coating antibody solution. Theplate was wrapped in parafilm and placed at 2-8° C. overnight.

On the next day, blocking solution was made by preparing Sigma gelatinat 1.5% w/v in carbonate coating buffer. The 96 well plate was removedfrom the refrigerator, residual binding sites in the plate wells wereblocked by adding 200 μl per well of the 1.5% w/v blocking solution. Theplate was placed at 37(±2)° C. for approximately 60 minutes.

ELISA wash solution (Phosphate buffered saline, 0.05% Tween 20; PBST)was prepared by dissolving one Fluka Phosphate buffered saline tablet in500 ml of purified water. Calibrator/sample diluent was prepared bydissolving Sigma gelatin at 1.0% w/v in PBST.

The calibration curve was prepared using Sigma IgG₁ Kappa with adeclared concentration of 1.0 mg/ml (96.8% purity). A 1/6667 dilution ofthe 1 mg/ml stock IgG₁ was prepared to give the top calibration standardof 150 ng/ml, this standard was serially diluted 1:1 with assay diluentto make the calibration series 150, 75, 38.5, 18.75, 9.375 ng/ml mouseIgG₁. Based on the protein concentrations for the purified antibodysamples (Sample 1—0.12 mg/ml, Sample 2—0.14 mg/ml), the samples werediluted 1/5000 for testing. The blocking solution was removed from theplate and the wells were washed once at 250 μl/well with assay diluent(PBST 1% gelatin). The calibration standards and media test samples wereapplied to the plate in triplicate at 100 μl/well. The plate was coveredwith parafilm, placed onto the plate shaker at a setting of 6/10 for 120minutes.

At the end of the sample incubation period, the standards and sampleswere removed and the plate was washed three times at 250 μl/well withassay diluent. The secondary antibody (goat anti mouse IgG conjugated tohorse radish peroxidase [HRP], Bethyl) was prepared at a 1/100,000dilution in assay diluent. The wash was removed and the secondaryantibody was added to the plate at 100 μl/well. The plate was coveredwith parafilm, and placed back onto the plate shaker at a speed of 6/10for a further 90 minutes.

At the end of the conjugate incubation, the secondary antibody solutionwas removed and the plate was washed three times with PBST buffer at 250μl/well, and then rinsed once with purified water. Tetra methylbenzidine (TMB) substrate (Sigma) was added to the plate wells at 100μl/well. A fresh piece of parafilm was used to cover the plate, whichwas incubated static at ambient laboratory temperature for 10 minutes.The enzyme-substrate reaction was terminated by adding 100 μl/well of 1Mphosphoric acid. The plates were read at 450 nM.

The A_(450/600) for both samples was just detectable above baseline butwell below the result for the 9.375 ng/ml calibration standard. TheMouse IgG₁ kappa ELISA correlates with the isotype result, both samplescontain very low concentrations of this isoform of the antibody.

Affinity of the expressed anti-hENT1 antibodies was demonstrated byELISA. The 10D7G2 monoclonal antibody was generated using a synthesizedpeptide sequence of the hENT-1 protein. A second ELISA was performed totest the affinity of the purified recombinant antibody for thissynthetic peptide sequence Ac-SKGEEPRAGKEESGVSC-amide (SEQ ID NO: 56).

Carbonate bicarbonate coating buffer was prepared as described for theIgG ELISA above. A vial of the synthetic peptide coupled to BSA(AC-SKGEEPRAGKEESGVSVSC-amide (SEQ ID NO: 59), MW: 1977, Mgs 0.25,coupled to BSA in 1 mg/l ml PBS) was reconstituted using 250 μl of thecarbonate bicarbonate coating buffer to give a theoretical concentrationof 1 mg/ml. This was further diluted 1/500 in the carbonate bicarbonateplate coating buffer to give 12 ml of coating solution at 2 μg/ml. AGreiner 96 well medium bind ELISA plate was coated at 100 μl per wellwith the 2 μg/ml BSA-peptide solution. The plate was wrapped in parafilmand placed at 2-8° C. overnight.

On the next day, blocking solution was made by preparing Sigma gelatinat 1.5% w/v in carbonate coating buffer. The 96 well plate was removedfrom the refrigerator, residual binding sites in the plate wells wereblocked by adding 200 μl per well of the 1.5% w/v blocking solution. Theplate was placed at 37(±2)° C. for approximately 60 minutes. PBST washbuffer and assay diluent (1% gelatin in PBST) were prepared as describedfor the IgG ELISA above. The 1/5000 dilutions of purified IgG samples 1and 2 were prepared as for the IgG sandwich ELISA described above. Theblocking solution was removed from the plate wells; the plate was washedonce at 250 μl/well with the assay diluent.

The test samples were applied to plate wells in triplicate at 100μl/well. The plate was covered with parafilm and placed onto the plateshaker (MPS1) at ambient temperature with shaking at 6/10 for 120minutes. At the end of the sample incubation, the samples were removedfrom the wells and the plate was washed three times at 250 μl/well withassay diluent.

The secondary antibody (goat anti mouse IgG conjugated to horse radishperoxidase [HRP], Bethyl) was prepared at a 1/100,000 dilution in assaydiluent. The wash was removed and the secondary antibody was added tothe plate at 100 μl/well. The plate was covered with parafilm, andplaced back onto the plate shaker at a speed of 6/10 for a further 90minutes. At the end of the conjugate incubation, the secondary antibodysolution was removed and the plate was washed three times with PBSTbuffer at 250 μl/well, and then rinsed once with purified water.

Tetra methyl benzidine (TMB) substrate (Sigma) was added to the platewells at 100 μl/well. The plate was covered with a fresh piece ofparafilm and was incubated static at ambient laboratory temperature for10 minutes. The enzyme-substrate reaction was terminated by adding 100μl/well of 1M phosphoric acid. The plates were read at 450 nM.

It is the results of the peptide ELISA that can be used to demonstratethe expression of functional anti peptide IgG by the transient CHOculture. To demonstrate the specificity of the peptide ELISA, 150 ng/mlSigma IgG1 kappa standard was added to wells D10-12, the results forthis sample are insignificantly different to the values recorded for thesix wells of assay diluent only (B10-12 & C10-12). Whilst theconcentration of the “negative control” was 5000 fold higher than thatof the test samples at 150 ng/ml, there is no risk that this wouldresult in a false negative (hook/prozone effect), 150 ng/ml is the topstandard used for the mouse IgG1 sandwich ELISA, which uses the sameplate type, sample diluent, conjugate wash solution and substrate. It isconcluded therefore that any signal generated by a test sample is theresult of binding to the peptide sequence only, and not any non specificinteraction of mouse IgG1 with the 96 well plate.

Although the signal generated by the two test samples were relativelylow they were sufficiently greater than the mouse IgG1 kappa control toconclude that both samples contain recombinant antibody with specificaffinity for the synthetic peptide sequence Ac-SKGEEPRAGKEESGVSC-amide(SEQ ID NO: 56).

In an attempt to generate a stronger positive result with the peptideELISA, 150 ml of conditioned (non-purified) media from a transientculture was used. Using an Amicon stirred cell model 8400 fitted with aYM 10 ultrafiltration disc, the 150 ml volume was reduced toapproximately 1.5 ml (100 fold concentration). The concentrated samplewas then tested using the peptide ELISA (method and materials asdescribed above) at dilutions of 1/10 to 1/640. The plate reader outputdemonstrated full functionality of the recombinant antibody expressedduring this transient culture against the synthetic peptide sequence.

The testing (isotype, mouse IgG₁ and peptide ELISAs and western blot)have demonstrated that the sequence of the variable region, derived fromthe 10D7G2 hybridoma cells has been accurately determined, transferredinto a plasmid construct and transiently expressed within a CHO hostcell line.

The sequences described herein have, therefore, been demonstrated tohave specific affinity for the synthetic peptide sequenceAc-SKGEEPRAGKEESGVSC-amide (SEQ ID NO: 56).

In the second study, the following heavy and light chain amino acidsequences derived from the 3A7C8 hybridoma cells were synthesized andcloned into the antibody expression plasmid shown in FIG. 5. The DNAcoding for the antibody light chain and heavy chain above wassynthesized and optimized for expression in CHO cells by Geneart(Germany). The DNA was inserted into a DHFR expression vector viarestriction sites BamHI and AvrII for the light chain and BglII and NheIfor the heavy chain. This vector can also be used for the development ofa stable cell line, by selection with Neomycin/G418 antibiotic and/ormethotrexate (a DHFR inhibitor).

Heavy chain amino acid sequence (SEQ ID NO: 57)

LYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVL HEGLHNHHTEKSLSHSPGKLight chain amino acid sequence (SEQ ID NO: 55)

KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The variable domains of the heavy and light chains are shown in bold inSEQ ID NO: 57 (heavy chain) and SEQ ID NO: 55 (light chain). Thecomplementarity determining regions (CDRs) are shown in boxes in SEQ IDNO: 54 (heavy chain) and SEQ ID NO: 55 (light chain). CDR sequences wereidentified using IMGT algorithms as described herein.

Several transfections will be carried out on the antibody expressionplasmid, under varying conditions in order to optimize the transfectionefficiency. For example, this plasmid will be tested using the transienttransfection methods described above.

Testing has been performed to confirm that the antibodies derived fromthe 3A7C8 hybridoma cells has been accurately determined, and furthertests, for example, the tests described above in connection with thesequences of the variable region derived from the 10D7G2 hybridoma cellswill demonstrate that the antibody expression plasmid shown in FIG. 5has successfully been transiently expressed within a CHO host cell line.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the descriptionand examples above are for purposes of illustration and not limitationof the following claims.

What is claimed is:
 1. An isolated monoclonal anti-human EquilibrativeNucleoside Transporter 1 (hENT1) antibody or antigen-binding fragmentthereof, wherein said antibody comprises: (a) a variable heavy chaincomplementarity determining region 1 (VH CDR1) sequence comprising theamino acid sequence GYTFTDYE (SEQ ID NO: 10); (b) a variable heavy chaincomplementarity determining region 2 (VH CDR2) sequence comprising theamino acid sequence IDPETGAI (SEQ ID NO: 11) or the amino acid sequenceIDPETGKT (SEQ ID NO: 40); and (c) a variable heavy chain complementaritydetermining region 3 (VH CDR3) sequence comprising the amino acidsequence TREFTY (SEQ ID NO: 12) or the amino acid sequence TRELTY (SEQID NO: 41); and (d) a variable light chain complementarity determiningregion 1 (VL CDR1) sequence comprising the amino acid sequenceQSLLFSNGKTY (SEQ ID NO: 24); (e) a variable light chain complementaritydetermining region 2 (VL CDR2) sequence comprising the amino acidsequence LVS (SEQ ID NO: 25); and (f) a variable light chaincomplementarity determining region 3 (VL CDR3) sequence comprising theamino acid sequence VQGTHFPWT (SEQ ID NO: 26), wherein said antibody orantigen-binding fragment thereof binds hENT1 or a biologically activefragment thereof.
 2. The antibody of claim 1, wherein said antibody orantigen-binding fragment thereof comprises a heavy chain variablesequence comprising an amino acid sequence selected from SEQ ID NO: 2,4, 6, 8, 9, 28, 30, 32, 34, 36, 38, 39, 51 and 53, and a light chainvariable sequence comprising the amino acid sequence selected from SEQID NO: 14, 16, 18, 20, 22, 23, 43, 45, 47 and
 49. 3. The antibody ofclaim 1, wherein said antibody is an IgG isotype or antigen-bindingfragment of an IgG isotype antibody.
 4. The antibody of claim 3, whereinsaid antibody is an IgG1 isotype or antigen-binding fragment of an IgG1isotype antibody.
 5. The antibody of claim 4, wherein said antibody isan IgG1 kappa isotype or antigen-binding fragment of an IgG1 kappaisotype antibody.
 6. An isolated monoclonal antibody or antigen-bindingfragment thereof comprising a heavy chain variable sequence comprisingthe amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 9, 28, 30, 32, 34, 36,38, 39, 51 or 53 and a light chain variable sequence comprising theamino acid sequence of SEQ ID NO: 14, 16, 18, 20, 22, 23, 43, 45, 47 or49, wherein said antibody or antigen-binding fragment thereof bindshuman Equilibrative Nucleoside Transporter 1 (hENT1) or a biologicallyactive fragment thereof.
 7. The antibody of claim 6, wherein saidantibody is an IgG isotype or antigen-binding fragment of an IgG isotypeantibody.
 8. A pharmaceutical composition comprising the antibody orantigen-binding fragment thereof of claim 1 and a carrier.
 9. Use of theantibody of claim 1 to detect a level of human Equilibrative NucleosideTransporter 1 (hENT1) expression or activity in a subject or patientsample.
 10. The use of claim 9, wherein the detected level of hENT1expression or activity in the subject or patient sample is used toidentify a course of treatment for the subject.
 11. The use of claim 10,wherein the course of treatment is administration of gemcitabine,gemcitabine-5′-elaidic acid ester, gemcitabine-N4-elaidic acid amide,cytarabine, cytarabine-5′-elaidic acid ester, ribavirin-5′-elaidic acidester, 5-azacytidine-5′-elaidic acid ester and combinations thereof. 12.The use of claim 9, wherein the detected level of hENT1 expression oractivity in the subject or patient sample is used to identify a subjector patient sample having low hENT1 expression or activity to identify acourse of treatment for the subject.
 13. The use of claim 12, whereinthe course of treatment is administration of gemcitabine-5′-elaidic acidester, gemcitabine-N4-elaidic acid amide, cytarabine,cytarabine-5′-elaidic acid ester, ribavirin, ribavirin-5′-elaidic acidester or 5-azacytidine-5′-elaidic acid ester.
 14. Use of the antibody ofclaim 1 as a companion diagnostic for a cancer drug or an antiviraldrug.
 15. The use of claim 14, wherein the cancer drug isgemcitabine-5′-elaidic acid ester, gemcitabine-N4-elaidic acid amide,cytarabine-5′-elaidic acid ester, ribavirin-5′-elaidic acid ester or5-azacytidine-5′-elaidic acid ester.
 16. A method of alleviating asymptom of a clinical indication associated aberrant human EquilibrativeNucleoside Transporter 1 (hENT1) expression or activity in a subject,the method comprising administering an antagonist of hENT1 to a subjectin need thereof in an amount sufficient to alleviate the symptom of theclinical indication associated aberrant hENT1 expression or activity.17. The method of claim 16, wherein said subject is a human.
 18. Themethod of claim 16, wherein said antagonist is a monoclonal antibody orantigen-binding fragment thereof.
 19. The method of claim 18, whereinsaid monoclonal antibody or antigen-binding fragment thereof comprises avariable heavy chain complementarity determining region 1 (VH CDR1)sequence comprising the amino acid sequence GYTFTDYE (SEQ ID NO: 10); avariable heavy chain complementarity determining region 2 (VH CDR2)sequence comprising the amino acid sequence IDPETGAI (SEQ ID NO: 11) orthe amino acid sequence IDPETGKT (SEQ ID NO: 40); and a variable heavychain complementarity determining region 3 (VH CDR3) sequence comprisingthe amino acid sequence TREFTY (SEQ ID NO: 12) or the amino acidsequence TRELTY (SEQ ID NO: 41), and a variable light chaincomplementarity determining region 1 (VL CDR1) sequence comprising theamino acid sequence QSLLFSNGKTY (SEQ ID NO: 24); a variable light chaincomplementarity determining region 2 (VL CDR2) sequence comprising theamino acid sequence LVS (SEQ ID NO: 25); and a variable light chaincomplementarity determining region 3 (VL CDR3) sequence comprising theamino acid sequence VQGTHFPWT (SEQ ID NO: 26), and wherein said antibodyor antigen-binding fragment thereof binds hENT1 or a biologically activefragment thereof.
 20. The method of claim 19, wherein said antibody orantigen-binding fragment thereof comprises a heavy chain variablesequence comprising an amino acid sequence selected from SEQ ID NO: 2,4, 6, 8, 9, 28, 30, 32, 34, 36, 38, 39, 51 and 53, and a light chainvariable sequence comprising the amino acid sequence selected from SEQID NO: 14, 16, 18, 20, 22, 23, 43, 45, 47 and
 49. 21. The method ofclaim 19, wherein said antibody is an IgG isotype or an antigen-bindingfragment of an IgG isotype antibody.
 22. The method of claim 21, whereinsaid antibody is an IgG1 isotype or antigen-binding fragment of an IgG1isotype antibody.
 23. The method of claim 22, wherein said antibody isan IgG1 kappa isotype or antigen-binding fragment of an IgG1 kappaisotype antibody.